Antibody Conjugated Lipid Nanoparticles: A New Targeted Drug Delivery Platform

Embedded within the rapid progress of nanomedicine technology, antibody-conjugated lipid nanoparticles (Ab-LNPs) are becoming a hotbed of precision medicine. Ab-LNPs technology possesses the distinct potential and market potential in drug delivery as well as an extended scope of possible uses in precision medicine.

What is an Antibody?

Antibodies are globular proteins made by B-lymphocytes that are specifically able to identify and associate with a protein or other biomolecule — the very fact that antibodies are useful for purification, detection and measurement of target molecules. Polyclonal or monoclonal antibodies can be generated by immunising animals like mice, rats, rabbits and goats. Often applied antibodies in research are also those conjugated to fluorescent or biotin dyes to show up and amplify signals in experiments. These antibodies are used in research, and because of their generality and specificity they are a necessity in biomedical research.

Antibody Conjugation

Antibody conjugation is a chemical linkage of antibodies to another object (eg, drug, nanoparticle or liposome). During the conjugation process, antibodies maintain their antigen-binding function but can also be made to deliver therapeutic drugs to target cells. This ensures that treatments are more specific and effective, with fewer systemic side effects. Antibody conjugation is performed in a few ways that are all very different in stability, efficacy, and usage. The most commonly used conjugation techniques include:

• Covalent Bonding: Linkers of chemical make stable covalent bonds between antibody and the conjugated molecule. Thiol-maleimide and NHS ester-amine links are, for example, popular links between antibodies and other targets.
• Non-Covalent Bonding: For this method, the antibodies are bound to surfaces like nanoparticles by weak interactions like electrostatic forces, hydrophobic or van der Waals forces. This approach offers freedom but is less stable than covalent conjugation.

What conjugation protocol you use will vary depending on the therapeutic aim, stability demands, and nature of target molecule.

Antibody Conjugated Lipid Nanoparticles

Antibody-conjugated lipid nanoparticles (Ab-LNPs) are a novel drug delivery system that allows for targeted delivery and drug uptake into infected cells or tissues by coating drugs on antibody-enhanced lipid nanoparticles; the effectiveness of these drugs is dramatically enhanced while protecting normal cells. Ab-LNPs are very intricately designed, and the structure of the LNPs, the properties of the antibodies and the number of antibodies on the surface of LNPs influence the efficacy of targeting, antigen binding, and the degree of internalization. From the first immunoliposomes , scientists began to try to glue antibodies to the surface of liposomes to make them more specific for cells they were targeting. This early study of immunoliposomes laid the foundation for the later technology of Ab-LNPs. As technology advanced, researchers developed a variety of coupling techniques and formulations that significantly improved the stability and targeting efficiency of Ab-LNPs. For example, Ab-LNPs targeting multiple receptors were developed for delivery of different types of therapeutic agents, including chemotherapeutic drugs, proteins, and gene therapy agents. The core advantage of Ab-LNPs lies in the fusion of two powerful therapeutic tools: antibodies and lipid nanoparticles.

Lipid Nanoparticles

Lipid nanoparticles are already established as an effective means of delivering hydrophobic and hydrophilic drugs, nucleic acids (such as mRNA, siRNA, and CRISPR components), and vaccines. Their biocompatibility, stability, and ability to encapsulate a wide range of therapeutic agents have made them a go-to platform for many drug delivery systems.

Antibody

However, without a targeting mechanism, these LNPs often suffer from a lack of specificity, resulting in poor cellular uptake and non-specific drug distribution. This is where antibodies play a pivotal role. By conjugating antibodies to the surface of lipid nanoparticles, the specificity of the antibody directs the nanoparticles to particular cells or tissues, where they can then deliver their therapeutic payload. This combination leverages the selective binding capacity of antibodies to specific cell surface antigens, ensuring that the drug or genetic material is delivered to the desired location.

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Antibody Conjugated LNP Function

The primary function of antibody-conjugated lipid nanoparticles is to enable targeted drug delivery. By leveraging the unique properties of antibodies, Ab-LNPs can:

• High Targeting Specificity: By incorporating antibodies that recognize specific antigens, Ab-LNPs can deliver drugs directly to diseased cells, reducing the risk of damage to healthy tissue.
• Improved Drug Efficacy: The ability to deliver drugs directly to the target site enhances therapeutic outcomes and improves bioavailability, especially for poorly soluble or unstable drugs.
• Reduced Systemic Toxicity: By concentrating the drug delivery at the target site, Ab-LNPs reduce the toxic side effects often associated with traditional treatments.
• Versatility: Ab-LNPs can be tailored for a wide range of therapeutic agents, including small molecules, proteins, nucleic acids (like siRNA or mRNA), and even vaccines.
• Scalability: The production of Ab-LNPs can be scaled up for clinical and commercial applications, with established methods for large-scale manufacturing.

Ab-LNPs thus offer a multi-faceted advantage in the treatment of diseases that require precise targeting, including cancer, genetic disorders, and infectious diseases.

How to Conjugate Antibody to LNP?

Conjugating antibodies to lipid nanoparticles (LNPs) is a critical step in the development of antibody-conjugated lipid nanoparticles (Ab-LNPs) for targeted drug delivery. The conjugation process ensures that the LNPs can bind to specific cell surface receptors, enabling the efficient and selective delivery of therapeutic agents. Several methods can be employed to conjugate antibodies to LNPs, each with its own set of advantages and considerations. Before diving into specific conjugation strategies, it is important to consider the following key factors:

Conjugation strategies for antibody conjugated nanoparticle (ACNP) generation. (Juan, A.; et al, 2020)

• Antibody Selection: The first step is selecting an appropriate antibody for conjugation. This will depend on the target antigen (e.g., cancer-specific markers or viral receptors). Antibodies can be fully human, monoclonal, or engineered fragments (like Fab, scFv) based on the required specificity, affinity, and pharmacokinetics.
• LNP Composition: The lipid nanoparticle formulation should be optimized for encapsulating the therapeutic payload (such as drugs, mRNA, or siRNA) while providing the necessary properties for antibody conjugation. Common lipids in LNPs include ionizable lipids (for transfection), phospholipids (for stability), and polyethylene glycol (PEG)-lipids (for circulation time). The lipid formulation must also support the correct orientation and retention of the conjugated antibody on the surface of the nanoparticle.
• Functionalization Sites: Conjugation should occur at specific sites on the antibody to preserve its binding affinity and activity. Typically, functional groups on the antibody (e.g., thiol groups, amines, or carboxyl groups) are targeted for conjugation.

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Covalent Conjugation - Crosslinker

Covalent conjugation using crosslinkers is one of the most commonly used methods to attach antibodies to LNPs. This strategy involves the use of bifunctional linkers that can react with both the antibody and a functional group on the lipid nanoparticle. This ensures a stable, long-lasting attachment.

• Amine-Reactive Crosslinkers: One popular approach involves using amine-reactive crosslinkers such as N-hydroxysuccinimide (NHS)-esters. The amine group on the antibody (typically located on the lysine residues) reacts with the NHS group on the crosslinker, which in turn binds to a carboxyl group on the lipid surface of the LNP.
• Thiol-Maleimide Chemistry: Another common covalent conjugation method is thiol-maleimide chemistry. This approach utilizes the thiol groups of cysteine residues in the antibody (either naturally present or engineered into the antibody) and maleimide-functionalized lipids to form a stable thioether bond.

Non Covalent Conjugation - Electrostatic Interactions

Non-covalent conjugation methods take advantage of electrostatic interactions between the negatively charged lipid nanoparticles and the positively charged regions of the antibody. This method is particularly useful for antibodies that have a significant number of basic amino acid residues (e.g., lysine or arginine).

• Functionalize the LNPs with anionic lipids (e.g., phosphatidylserine or negatively charged PEG-lipids).
• Incubate the LNPs with the positively charged antibody in an appropriate buffer.
• Electrostatic interactions will drive the association between the antibody and the LNP surface.

Click Chemistry Antibody Conjugation

Click chemistry, particularly azide-alkyne cycloaddition, offers an efficient and selective method for conjugating antibodies to lipid nanoparticles. The "click" reaction allows for highly specific conjugation without the need for toxic reagents or complex purification steps.

• Functionalize the antibody with an alkyne or azide group.
• Modify the LNPs with the complementary azide or alkyne group (via a lipid linker).
• Mix the antibody and LNPs in the presence of a catalyst to induce the "click" reaction, forming a stable covalent bond.

Biotin Streptavidin Conjugation

Biotin-streptavidin interactions provide another method for antibody conjugation, particularly when a more flexible, reversible attachment is desired.

• Functionalize the antibody with biotin (via either direct labeling or through a biotinylation reagent).
• Modify the LNPs with streptavidin or avidin.
• Biotin-streptavidin binding will tether the antibody to the LNP surface.

Applications of Antibody Conjugated Lipid Nanoparticles

Targeted Cancer Therapy

One of the most promising applications of Ab-LNPs is in targeted cancer therapy. Cancer cells often overexpress specific surface markers that can be recognized by antibodies. By conjugating antibodies that target these tumor-associated antigens (TAAs) to lipid nanoparticles, it becomes possible to direct cytotoxic drugs, small molecules, or RNA-based therapeutics specifically to cancer cells, thereby sparing healthy tissues and reducing systemic toxicity.

Gene Therapy and RNA Delivery

Ab-LNPs have emerged as a powerful tool for gene therapy and the delivery of RNA-based therapeutics, including mRNA, small interfering RNA (siRNA), and messenger RNA (mRNA) vaccines. By conjugating antibodies that recognize cell surface receptors, Ab-LNPs can facilitate the targeted delivery of genetic material to specific cell types, increasing the therapeutic effectiveness of gene-editing techniques and RNA therapies.

Immunotherapy and Immune Modulation

Antibody-conjugated lipid nanoparticles are also increasingly being explored in immunotherapy, particularly in the context of checkpoint inhibitors, CAR-T cell therapy, and immune modulating drugs. These therapies aim to manipulate the immune system to target and destroy diseased cells, especially cancer cells, by enhancing immune recognition or stimulating immune responses.

Vaccine Development and Delivery

Ab-LNPs are also being investigated as a vehicle for delivering antigenic vaccines. The ability of LNPs to deliver RNA, proteins, or peptides combined with the targeting capability of antibodies enables the development of highly effective vaccines. These vaccines can be aimed at specific pathogens or cancer cells, providing a more targeted and efficient immune response.

Diagnostic Imaging and Theranostics

Ab-LNPs are also playing an increasing role in diagnostic imaging and theranostic applications, where the same agent is used for both therapeutic and diagnostic purposes. The specificity of antibodies allows for the precise targeting of biomarkers that are overexpressed on the surface of tumors or infected cells.

Antibody-Conjugated LNPs for Ocular Drug Delivery

The eye is a challenging organ to treat due to its unique anatomical and physiological barriers, including the blood-retinal barrier. Ab-LNPs are being explored for the targeted delivery of ocular therapeutics, including gene therapies, small molecules, and biologics. By using antibodies that target specific receptors expressed in ocular tissues, these nanoparticles can provide localized drug delivery to treat retinal diseases, such as macular degeneration or diabetic retinopathy.

Reference

1. Juan, A.; et al. An Overview of Antibody Conjugated Polymeric Nanoparticles for Breast Cancer Therapy. Pharmaceutics. 2020, 12 (9): 802.